The sex disparity in the incidence and progression of cardiovascular disease (CVD), including peripheral arterial disease (PAD), is a troubling clinical observation. It is well known that CVD manifests differently in men and women, with more women than men suffering from the disease. Further, within the vascular network, there are mechanical microenvironments that favor CVD development, for example, atherosclerotic plaques preferentially form at regions of disturbed flow and low fluid shear stresses such as, vasculature bifurcations. Yet, no reports have correlated these two separate observations at the cellular level, specifically, the sex differences in CVD and local vascular mechanics. This project aims to 1) assess the functional changes in male and female human umbilical vein endothelial cells in response to combined physiological fluid shear stresses and substrate stiffness by morphometric, secretory, and genetic analyses, 2) assess the functional changes in male and female human aortic smooth muscle cells in response to combined physiological cyclic stretching and substrate stiffness by morphometric, mechanical, and genetic analyses, and 3) correlate the disparate functional response of male and female vascular cells using statistical methods. Upon completion of these aims, a foundational understanding of vascular cell functionality in vitro as it pertains to sexual dimorphism in complex mechanical microenvironments will be gained. A factorial design of experiments will be used to systematically assess the influence of cell sex, laminar fluid shear stress or cyclic stretch, and the underlying substrate stiffness. The effects of the mechanical microenvironment will be assessed using well-defined flow and stretch bioreactors with RGD-conjugated polyacrylamide gel substrates. Cell morphology will be quantified by immunofluorescence microscopy methods. Glycocalyx, vasoregulatory, and estradiol secretory products will be quantified by commercial assays. Cellular mechanical properties will be measured by atomic force microscopy. The regulation of a large set of vascular cell genes including a subset related to CVD will be measured by quantitative reverse transcription polymerase chain reaction. Statistical analyses will be performed to relate gene expression results to morphological, secretory, and mechanical responses and then to correlate them to a given sex and mechanical force combination. This work follows the 2015 NIH mandate to consider sex as a biological variable by investigating the clinical observations of the sex disparity in cardiovascular disease at the cellular level. The significance for this work is that future imaging modalities that quantify vascular forces such as with 4D MRI, will be able to indicate areas within the vasculature that are susceptible to localized cellular dysfunction using correlative statistical models and ultimately predict a patient's location-specific vulnerability for disease. This proposal is part of a predoctoral training fellowship at the University of Florida. The trainee will learn to conduct the experiments proposed and to grow professionally from the guidance of a comprehensive mentoring team of clinicians, cell and molecular biologists, computational theorists, and material scientists.